This proposal seeks to identify and characterize drug-like molecules that inhibit ciliary Hedgehog (Hh) signaling. Cells orchestrate their behaviors by communicating through secreted signals such as Hh proteins. Unlike other intercellular signals, mammalian Hh proteins are transduced through primary cilia, microtubule-based projections on the surface of many cells. The Reiter lab discovered that a central component of the Hh pathway, Smoothened (Smo), moves to the primary cilium in response to Hh stimulation where it activates the downstream pathway. The cilium is essential for Hh signaling both in embryonic development and in oncogenesis. Activating mutations in Smo cause basal cell carcinoma (BCC), the most common cancer in the U.S., and medulloblastoma, the most common solid cancer in children. A Smo antagonist was recently approved for clinical use, although cancers can become resistant quickly. Smo antagonists that act through complementary mechanisms may prevent the emergence of resistance in Hh-associated cancers. To identify novel Hh pathway antagonists and gain insight into ciliary function, the Reiter and Arkin labs completed a pilot screen for inhibitors of Smo movement to cilia. This screen identified novel inhibitors, some of which block BCC cell growth by inhibiting Smo translocation and some of which block ciliogenesis. Investigating the mechanisms by which these inhibitors act revealed previously unknown aspects of ciliary Hh signaling. This project will expand the screen, identify the molecular mechanisms underlying inhibitor action, and understand ciliary signaling. Specifically, we will: 1) screen in-house and Chemical Biology Consortium libraries, substantially expanding the chemical space that has been assessed, 2) execute secondary screens to efficiently identify inhibitors that act through previously undescribed mechanisms, 3) use newly identified antagonists, together with unique genetic tools, to uncover unrecognized steps of ciliogenesis and Smo translocation. Despite their importance to both development and disease, the mechanisms underlying ciliogenesis and Hh signaling remain unclear. The proposed investigation will provide tools to reveal how cells communicate through cilia. These compounds may also provide leads for novel chemotherapeutics, critical for preventing the emergence of resistance and relapse in Hh pathway-associated cancers.